Chronic hepatitis C (HCV) infections are common in alcoholics with or without liver disease contributing to significant morbidity and mortality. The reasons for high rates of persistent viral hepatitis infection are unknown but may partially relate to the effects of alcohol on the humoral and cellular immune response to viral structural and nonstructural proteins. We established an animal model system of HCV specific antibody stimulation, CD4+ proliferative response, and CD8+ CTL activity in the context of genetic immunization to test this hypothesis. We plan to do the following: Specific Aim #1 - Further establish the differential effects of ethanol on the immune response(s) to HCV structural and nonstructural proteins in the context of genetic immunization with respect to: a. Assess variations in CD4+ T cell proliferative activity particularly against HCV core, NS3, NS3 helicase, NS4, NS5A and NS5B proteins. b. Evaluate Th1 and Th2 responses to determine if ethanol consumption shifts the immune response from the Th1 to Th2 phenotype. c. Characterize CD8+ CTL precursor frequency and function including possible differential effects of ethanol at the epitope level. d. Examine CTL activity in vivo against HCV derived peptides using an animal model of tumor growth. e. Determine the duration and reversibility of ethanol effects on CD4+ and CD8+ T-cell responses by crossover feeding studies with an isocaloric control diet. Specific Aim #2 - Develop approaches to augment and/or amplify humoral and cellular immune responses generated by DNA immunization in the setting of chronic ethanol administration, a. Explore whether systemic administration (murine and human IFN-a2, or pegylated IFN- a2) is more effective than local administration of cytokine expression construct (IL-2, GM-CSF, IL-12) at the site of gene delivery with respect to augmentation of the humoral and cellular immune response(s) to HCV structural/nonstructural proteins. b. Determine the value of co-immunization with CpG motifs as an enhancer of the host B and T cell immune responses. c. Employ chimeric constructs to assess their possible role as a stimulant of virus specific B and T cell activities. Thus, principle long term goals of this application are to develop: a clinical DNA vaccine approach, effective alone or in combination with other antiviral agents (IFN-2a, etc.), to enhance the host immune response in an attempt to eradicate persistent viral infection from the liver in alcoholics; and obtain a clearer understanding of ethanol's effects on the immune response to HCV structural and nonstructural proteins in an experimental animal model system thereby generating principles applicable to chronic alcoholics